ABSTRACT
Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.
Subject(s)
Leishmania mexicana/immunology , Leishmaniasis, Cutaneous/immunology , Protozoan Vaccines/immunology , Toll-Like Receptor 9/immunology , Administration, Intranasal , Animals , Antigens, Protozoan/immunology , CpG Islands , Dendritic Cells/immunology , Dendritic Cells/parasitology , Extracellular Traps , Interferon-gamma/immunology , Macrophages/immunology , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/parasitology , Nitric Oxide/biosynthesis , Parasite Load , Toll-Like Receptor 9/genetics , VaccinationABSTRACT
O timo é um órgão linfóide central onde precursores derivados da medula óssea sofrem um complexo processo de maturação que ocorre à medida que os timócitos migram no microambiente tímico, uma rede tridimensional composta de diferentes tipos celulares. Estas células microambientais interagem com timócitos, provendo sinais que regulam o desenvolvimento intratímico normal de células T. Estes sinais são liberados em nichos do microambiente tímico, nos quais um determinado tipo celular apresenta um padrão de sinalização específico, e influenciam determinados estágios específicos de diferenciação/migração dos timócitos. Em nosso trabalho, identificamos genes diferencialmente expressos entre células epiteliais tímicas corticais e medulares. Alguns genes fazem parte da regulação neuroendócrina do epitélio tímico, como por exemplo, timopoietina e receptores hormonais; outros, VCAM e fibronectina, participam da adesão e migração de timócitos no microambiente tímico. A identificação de tais genes contribui para o entendimento dos sinais fornecidos por nichos microambientais aos timócitos em desenvolvimento. Estes sinais geram a comunicação timócito/microambiente que é bilateral (conceito de cross-talk), o que implica que a interação com o timócito deva modular a expressão gênica no epitélio tímico. De fato, experimentos de microarranjos de DNA mostraram que tal interação gerou ma modulação de 114 genes nas células epiteliais corticais, 142 genes nas células medulares de camundongo e ainda, mais de 350 genes em células epiteliais humanas. Para aprofundar os conhecimentos sobre as interações timócito/epitélio tímico mediadas por proteínas de matriz celular (ECM) e seus receptores, fizemos o silenciamento do gene codificante para a molécula CD49e (cadeia alfa-5 de um dos receptores de fibronectina, a integrina VLA-5) nas células epiteliais tímicas humanas, através de interferência de RNA (RNAi). Vimos que o silenciamento deste gene modula a expressão gênica de...timócitos.
Subject(s)
Epithelial Cells , Extracellular Matrix , Gene Expression , Integrins , RNA Interference , Thymus GlandABSTRACT
We screened the TP53 gene for mutational status in 40 breast tumor cases by polymerase chain reaction, single-strand conformational polymorphism, and gene sequencing. Many mutations of this gene have been described in specific databases. In our study, a new T-->C point mutation was identified in intron 6 at position 13989 in a grade III medullary ductal carcinoma. Other variations in intron 6 have been described in patients with Li-Fraumeni syndrome. One of these variations was reported to inhibit apoptosis and prolong cell survival, thereby increasing breast cancer risk. Nevertheless, more studies are necessary to establish whether this mutation has a role in breast cancer risk.
